Critical events in HIV disease occur in lymphoid tissue where HIV kills infected CD4+ cells as well as many non-infected cells, disrupting a complex system of cell-cell interactions that mediate normal immune response. The general aim of the project is to determine the underlying mechanism(s) of HIV-1-mediated immunopathogenesis using our system of ex vivo-infected human tonsils. In vivo the ability of HIV-1 to use CXCR4 coreceptor exclusively or in addition to CCR5 coreceptor is often associated with massive loss of CD4+ T cells and rapid disease progression. The working hypothesis we are currently testing is that the HIV-1 env gene that determines the usage of CXCR4, exclusively or in addition to CCR5, is sufficient to induce T cell depletion and immunosuppression. Also, we investigate the contribution of HIV accessory genes, in particular that of nef gene to viral tissue cytopathicity. Earlier we showed that coreceptor specificity determines cytopathicity of mono-tropic HIV-1 variants of B subtype. This year we investigated (i) whether co-receptor specificity determinines cytopathicity of HIV-1 variants of subtypes other than B; (ii) whether the cytopathicity of dual-tropic R5X4 HIV-1 variants of B subtype is determined by their preferential usage of a particular co-receptor, and (iii) whether the contribution of an HIV accessory gene nef to HIV pathogenesis is associated with modulation of cell surface receptors. To investigate the ex vivo cytopathicity of R5 and X4 HIV-1 variants of non-B subtypes, we used primary isolates of subtypes A, E, and C and prototypic subtype B variants. We observed no relationship between the maximal level of viral replication and either coreceptor usage or viral subtype. However, we did observe a strong correlation between viral coreceptor usage and CD4+ T lymphocyte depletion across all subtypes tested. All R5 HIV-1 infections resulted in mild depletion of CD4+ T cells, whereas all X4 HIV-1 infections caused severe depletion of CD4+ T cells irrespective of their subtype. Thus, non-B HIV-1 subtypes are similar to HIV-1 subtype B in that coreceptor specificity determines cytopathicity in human lymphoid tissue infected ex vivo. To find out whether differential cytopathicity of R5X4 viruses is also related to their preferential use of a particular coreceptor, we infected CCR5 delta32 tissue with three R5X4 HIV-1 variants: 89.6, 89-v345.FL, and 89-v345.SF; the latter two are isogenic to 89.6 except for the V3-V5 region of gp120. Variant 89.6 severely depletes tissue of CD4+ T cells, whereas 89-v345.SF depletes only mildly. Variant 89-v345.FL was an intermediate depletor. Both in CCR5 delta 32 and in normal lymphoid tissue, 89.6 and 89-v345.FL replicated with similar kinetics. In contrast, 89-v345.SF did not replicate in lymphoid tissue lacking CCR5. In agreement with these results, replication of 89.6 was completely inhibited by an CXCR4 ligand AMD3100 but was insensitive to a CCR5 ligand RANTES, whereas 89-v345.SF replication was almost completely inhibited by RANTES but not by AMD3100. Replication of 89-v345.FL was reduced both by AMD3100 and by RANTES. Among two studied primary isolates, which were dual-tropic in transfected cell lines, one was not inhibited by RANTES but was almost completely blocked by AMD3100. Thus, in lymphoid tissues some R5X4 isolates appear to utilize CXCR4 exclusively, whereas others use CCR5 exclusively. R5X4 HIV variants that use CXCR4 preferentially are highly cytopathic, whereas those preferentially using CCR5 deplete CD4+ T cells mildly. Hence, preferential coreceptor usage determines cytopathicity of R5X4 variants. Cell surface receptors may mediate the effects of an accessory gene nef on HIV infection. Tissues were infected with NL4-3 clones bearing nef alleles from sequential patient's isolates, as well as with clones bearing recombinant alleles. The HIV-1 variants containing the nef alleles isolated at later time points showed two- to-three fold higher levels of replication than those containing the nef alleles initially isolated at the early time point. Higher levels of replication correlated with efficient depletion of CD4+ T cells. There was a significant correlation between Nef activity in CD4 down-regulation and the efficiency of virus replication in ex vivo-infected tissue, suggesting a link between Nef-mediated CD4 downregulation, viral replication, and disease progression. In summary, coreceptor preference of HIV-1 variants is a sufficient determinant of viral cytopathicity for monotropic viruses of B and non-B subtypes as well as for dual-tropic viruses; and down-modulation of cell surface receptors may mediate nef-effects on HIV-1 infection in lymphoid tissue.